CFTR AND ALIMENTARY EPITHELIAL ACID/ BASE TRANSPORT

CFTR 和消化道上皮酸/碱转运

• 批准号: 2458862
• 负责人: LANE L CLARKE
• 金额: $ 10.2万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2458862
• 关键词: acid base balance; bicarbonates; cell morphology; chloride channels; cyclic AMP; cystic fibrosis; gastrointestinal absorption /transport; gastrointestinal epithelium; gene deletion mutation; genetically modified animals; immunologic assay /test; intestinal mucosa; ion transport; laboratory mouse; membrane transport proteins; microelectrodes; protein structure function; tissue /cell culture; voltage /patch clamp

Gastrointestinal disease manifestations in the human genetic disease, cystic fibrosis (CF), are indicative of the central role that the cystic fibrosis transmembrane conductance regulator (CFTR) plays in gastrointestinal electrolyte transport physiology. Meconium ileus/distal obstruction syndrome, maldigestion/malabsorption disorders, and an increased risk of gastroduodenal ulcer disease and neoplasia have been reported for patients with mutations in the cftr gene and, hence, its defective epithelial protein product, CFTR. Although it is now widely recognized that CFTR can function as a cAMP-activated Cl- channel protein, numerous reports have described significant effects of CFTR mutations on other epithelial transport processes, including Na+ absorptive mechanisms, transcellular bicarbonate movements and sulfate transport. Our preliminary findings in a CFTR "knockout" mouse model of CF demonstrate that CFTR is critical for intestinal transcellular pH regulation by being required for 1) cAMP-stimulated HCO3- secretion in the proximal duodenum, and 2) cAMP-induced inhibition of coupled Na+/H+, CL0/HCO3- exchange mechanisms in the jejunum. Furthermore, the reported location of these transport mechanisms suggest that CFTR expression is not only localized to the intestinal crypts but uniquely functions in villous epithelium. The long-term goal of this laboratory is to uncover the cellular and molecular mechanisms involved in the modulation of intestinal acid/base transporters by CFTR. Littermates of the CFTR ~knockout" mouse model will be used to study intestinal epithelia with normal [cftr(+/+) mice], reduced [cftr(+/-) mice], or absent [cftr(-/-) mice] CFTR protein expression. In Specific Aim 1, we will identify the role of CFTR in cAMP-induced HCO3- transport in the proximal duodenal mucosa. This aim will be accomplished using voltage-clamp measurement of isolated transepithelial ion currents and immunodetection of CFTR protein in epithelial cell lysates. In Specific Aim 2, we will localize CFTR (or other anion conductances) and relevant intracellular anion driving forces along the crypt-villus axis using intracellular ion- selective microelectrode recordings. Isolated intact villous epithelium will be used for immunodetection of CFTR and to localize relevant ionic currents. In Specific Aim 3, we will identify the role of CFTR in cAMP- mediated inhibition of coupled Na+/H+, Cl-/HCO3- exchange mechanisms in the intestine. Isotopic flux and cell volume measurements will be used to test the hypothesis that CFTR-dependent changes in cell volume inhibit the coupled Na+/H+, Cl-/HCO3- absorptive process. Reconstitution of CFTR-negative alimentary epithelial cell lines with wild-type CFTR will be used to verify or disprove the hypothesis.

胃肠道疾病是人类遗传性疾病的表现， 囊性纤维化（CF），表明囊性纤维化的核心作用， 纤维化跨膜传导调节因子（CFTR）在 胃肠电解质转运生理学胎粪性肠梗阻/远端 阻塞综合征、消化不良/吸收不良疾病，以及 胃十二指肠溃疡疾病和肿瘤的风险增加， 报告了cftr基因突变的患者，因此， 缺陷型上皮蛋白产物。虽然现在广泛 认识到CFTR可以作为cAMP激活的Cl-通道起作用， 许多报道已经描述了CFTR的显著作用， 其他上皮转运过程的突变，包括Na+ 吸收机制，跨细胞碳酸氢盐运动和硫酸盐 运输我们在CFTR“敲除”小鼠模型中的初步发现， CF表明CFTR对肠道跨细胞pH至关重要 1）cAMP刺激的HCO 3分泌所需的调节， 近端十二指肠，和2）cAMP诱导的抑制耦合Na+/H+， 空肠中的CL 0/HCO 3交换机制。此外，据报道， 这些转运机制的定位表明，CFTR表达是 不仅局限于肠隐窝， 绒毛上皮这个实验室的长期目标是 参与调节的细胞和分子机制 肠酸/碱转运蛋白。CFTR的同窝仔 “基因敲除”小鼠模型将用于研究肠上皮细胞， 正常[cftr（+/+）小鼠]、减少[cftr（+/-）小鼠]或不存在[cftr（-/-） 小鼠] CFTR蛋白表达。在具体目标1中，我们将确定 CFTR在cAMP诱导的十二指肠近端HCO 3转运中的作用 粘膜这一目标将通过电压钳测量来实现 分离的跨上皮离子电流和CFTR的免疫检测 上皮细胞裂解物中的蛋白质。在具体目标2中，我们将 CFTR（或其他阴离子电导）和相关细胞内阴离子 驱动力沿着隐窝绒毛轴使用细胞内离子- 选择性微电极记录分离的完整绒毛上皮 将用于CFTR的免疫检测并定位相关离子 电流。在具体目标3中，我们将确定CFTR在cAMP中的作用。 介导的Na+/H+，Cl-/HCO 3-交换机制的抑制， 肠子将使用同位素通量和细胞体积测量 为了检验CFTR依赖性细胞体积变化的假设， 抑制Na ~+/H ~+、Cl ~-/HCO ~（3-）的耦合吸收过程。复溶 具有野生型CFTR的CFTR阴性消化上皮细胞系 将被用来验证或反驳假设。